The visual pigment rhodopsin is a constitutively active lipid scramblase capable of moving phospholipids rapidly between the leaflets of a membrane bilayer. This novel activity of rhodopsin plays a key role in enabling the ABC transporter ABCA4 to prevent accumulation of retinaldehydes and mitigate the formation of Vitamin A dimers. The toxic buildup of these molecules in the retina is considered to contribute to mechanisms underlying retinopathies such as Stargardt's disease and age-related macular degeneration. Both rhodopsin and its apo-protein opsin are scramblases, and here we are interested in understanding the molecular mechanism by which they translocate phospholipids. Based on preliminary results from atomistic molecular dynamics simulations we hypothesize that lipids are transported at the interface of specific transmembrane helices in opsin. Plasma membranes lack constitutive phospholipid scramblase activity, and thereby sequester the signaling lipid phosphatidylserine (PS) in the inner leaflet until scramblases are activated. However, cells that over-express opsin unexpectedly do not display PS at their surface. We hypothesize that high cholesterol levels in the plasma membrane silence opsin's scramblase activity. We propose two specific aims to test these hypotheses via biophysical, biochemical and computational methods. In the first aim we will combine experimental and in silico analyses to test the lipid translocation pathway within opsin predicted by our molecular dynamics simulations. Using site-directed mutagenesis we will crosslink transmembrane helices and also alter the pathway environment in order to disrupt transport, as measured in our fluorescence-based scramblase assays. We will also analyze the mutant opsins by molecular dynamics simulations in order to obtain structural context and mechanistic insights into the experimental results. In the second aim, we will use cell and vesicle-based assays to determine the effect of cholesterol on lipid scrambling, in conjunction with molecular dynamics simulations of wild-type opsin in cholesterol- containing membranes. The proposed studies will provide the first insights into the mechanism of lipid scrambling. As scramblases have only recently been discovered, their molecular mechanism is unknown and the field is in its infancy. The knowledge that we gain here will set the stage for future elucidation of this process in molecular detail. This has important implications for physiology, because of the role that opsin's scramblase mechanism is likely to have in supporting the flippase activity of ABCA4 in photoreceptor disc membranes. Deficiencies in this mechanism are clearly associated with retinopathies. Moreover, because opsin's scramblase activity is shared by other G protein-coupled receptors (GPCRs) such as the ?2-adrenergic receptor, our experiments also have the potential to open a new area of GPCR biology.